In animals, the seven enzymes involved in do novo fatty acid synthesis from malonyl-CoA are integrated into two polyfunctional polypeptides. The growing acyl chain is translocated on a mobile 4'-phosphopantetheine arm repeatedly through sites of condensation, ketoreduction, dehydration and enoyl reduction, ultimately to the site of chain termination. The animal fatty acid synthetase offers a unique opportunity for evaluating possible advantages of a polyfunctional polypeptide system of organization over a collection of separate, discrete enzymes, for the chain-terminating reaction can be catalyzed with equal efficiency either by one of the covalently-linked domains, thioesterase I, or by a separate medium-chain-teminating enzyme, thioesterase II (e.g. in mammary and uropygial glands), which is not a structural component of the fatty acid synthetase. One aim of this program is to map the domain structure of the mammalian fatty acid synthetase both linearly, using limited proteolysis as a dissecting tool and a combination of affinity labels, antibodies and partial enzyme activities as specific domain markers, and spatially, using intra and inter-chain cross-linking. A structural basis will be sought to account for the ability of ruminant (but not non-ruminant) fatty acid synthetases to utilize and synthesize medium-chain acyl-CoAs. A novel approach is described to determine whether the two subunits of the mammalian fatty acid synthetase function symmetrically, i.e. each simultaneously elongating an acyl moiety, or asymmetrically, assembling a single acyl moiety per dimer. The mechanism of action of the two types of chain-terminating enzymes will be compared by exploiting reagents, of both the monofunctional and bifunctional type, which attack specifically either the active site region or a putative interfacing region, which facilitates interaction with the fatty acid synthetase. By analysis for amino acid sequency homology of the medium-chain-terminating thioesterases of mamary and uropygial glands, we will explore the possibility that these modified skin glands may have some common evolutionary ancestry.